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Biofilm inhibiting phytometabolites
Abstract
Biofilms are produced by some microorganisms to attach to any surface. These layers are mainly in exopolysaccharide (EPS) nature, but also contain water, protein, and nucleic acids. One of the most important functions of this matrix is to protect microorganisms against drastic changes in some environmental factors such as UV light, water activity, osmotic pressure, pH, radiation, and the presence of antimicrobial agents. Being one of the main reasons for several diseases, which affect public health, biofilm is also responsible for several significant issues in livestock, agriculture, and food industries, and also in ventilation and irrigation systems.
The most common procedures used for preventing formation or removing biofilms completely, which were already formed, include limiting nutrients required for microbial growth, sterilization of surface materials to prevent bacterial attachment, or removing biofilms by chemical or mechanical treatments. However, microorganisms having the ability to form biofilms exhibit different behaviours from other microorganisms in terms of their growth rates and resistance to antibiotic or antifungal treatments. Besides, increasing resistance to antibiotics or antifungal agents in biofilm-forming microorganisms leads to remarkable problems in preventing biofilm formation or removing biofilms, which are already formed. Thus, scientists conducting researches for discovering novel agents, which can be used against both inhibiting biofilm formation and removing it. Researches presented that several phytometabolites have a great potential of being used as anti-biofilm agents.
This chapter aims to present some detailed information about biofilms and to review the researches about the potential of phytometabolites as anti-biofilm agents.
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Plant-derived molecules are excellent alternatives to antibiotics as anti-infective agents owing to their minimal cytotoxicity. Herein, the anti-infective property of the hydroxyflavone baicalin, was investigated against biofilms of the key dental caries pathogen Streptococcus mutans. Baicalin inhibited sucrose-dependent biofilm formation at a concentration of 500 µg ml⁻¹ without affecting bacterial growth. It significantly inhibited acid production for an extended period of 8 h. Microscopic analysis revealed a 6-fold reduction in the number of adhered cells with baicalin treatment. Transcriptomic analysis of the mid-log phase and biofilm cells showed marked downregulation of the virulence genes required for biofilm formation and acid production. This study sheds significant new light on the potential for baicalin to be developed into an anti-caries agent.
Syringopicroside is a natural drug with antibacterial activity, which is the main ingredient of Syringa oblata Lindl (S. oblata). In order to further develop the application of S. oblata and evaluate the ability of syringopicroside against Streptococcus suis (S. suis), this investigation first applied an ultrasonic-assisted method to extract syringopicroside, and then response surface methodology (RSM) was performed to get the optimum condition. Based on RSM analysis, a second-order polynomial equation about the syringopicroside yield and four variables, including ultrasonic power, time, temperature, and liquid-to-solid ratio, was purposed. Through RSM prediction and model verification experiments, the optimum conditions were determined, as follows: ultrasonic time was 63 min, temperature was 60 °C, a liquid-to-solid ratio was set to 63 mL/g, and ultrasonic power was 835 W. Under this condition, a high syringopicroside yield was obtained (3.07 ± 0.13 mg/g), which was not significantly different with a predicated value. After separation and purification by HPD 500 microporous resin, then mass spectrum was applied to identify the main ingredient in aqueous extract. A minimal inhibitory concentration (MIC) assay revealed the value against S. suis of syringopicroside was 2.56 µg/µL and syringopicroside with sub-inhibitory concentrations that could effectively inhibit biofilm formation of S. suis. Besides, scanning electron microscopy analysis indicated syringopicroside could destroy the multi-layered aggregation structure of S. suis. Finally, molecular docking analysis confirmed that syringopicroside was combined with Orfy protein of S. suis through hydrogen bonds, hydrophobic interaction, and π-π stacking.
Background
: Biofilms are responsible for the growing resistance of bacteria to antibiotics, preventing the antibiotic accessibility to bacterial cells. As an alternative to antibiotics, fatty acids (FAs) have been explored for their antibiofilm activities due to their diffusible nature and ability to modulate the membrane fluidity of the bacteria. Fatty acids have been shown as anti-biofilm agents mostly for Gram-positive pathogens. Therefore, we chose to explore the anti-biofilm activity of FAs from Withania somnifera (L.) Dunal (Solanaceae) seeds fixed oil (WSSO) on the Gram-negative pathogen, Salmonella enterica. Biofilm formation in Salmonella spp enhances its resistance to antibiotics and host immune response, with consequent increase in its virulence and chronicity of infection. FAs from WSSO was reported for their effectivity against Psoriasis-like skin inflammation. Biofilms are implicated in Psoriasis pathology and anti-biofilm therapy can provide new treatment options. In view of its biological importance, we have explored the activity of WSSO against planktonic and biofilm forms of S. enterica.
Purpose
: The purpose of the current study is to evaluate the potentials of fixed fatty acids from an important medicinal plant (W. somnifera) as antibiofilm agents against a Gram-negative bacterium (S. enterica).
Methods
: Antibacterial activity of WSSO against planktonic form of S. enterica was evaluated through broth microdilution method. Antibiofilm activity in terms of prevention of biofilm initiation, inhibition of biofilm formation and disruption of mature biofilm were quantified through crystal-violet staining. WSSO induced loss of membrane integrity, and concomitant effect on S. enterica motility were assessed through quantification of intracellular potassium and nucleotide effluxes, and motility assays, respectively.
Results
: The minimal inhibitory concentration of WSSO required for 50 % reduction in the planktonic bacterial load (IC50) was 5.78 mg/ml. Cells lost their motility when treated with WSSO at IC50. At a similar concentration of 6.28 mg/ml, WSSO disrupted mature biofilm of S. enterica. Bacterial cell membrane was compromised after treatment with 5.40 mg/ml WSSO as evident from potassium (K⁺) ion and nucleotide effluxes.
Conclusion
: WSSO not only prevented initiation of biofilm formation and but also efficiently disrupted mature biofilm of S. enterica. Taken together, these results indicated that WSSO has the potentials to be used as an alternative antibacterial agent against Gram-negative pathogenic S. enterica. The mode of action of WSSO seems to be linked to its ability to modulate membrane integrity and to restrain bacterial motility, most likely by its abundant and trace fatty acid contents.
Background and objectives:
Escherichia coli and some Salmonella serovars cause various disease manifestations in poultry leading to significant economic losses. The widespread and imprudent use of antibacterial agents in poultry flocks have increased resistant to many antibacterial agents which has become a major public health concern. Some medicinal plants may be alternative to antibacterial agents. The purpose of this study was to investigate the antibacterial and anti-biofilm activity of summer savory essential oil against E. coli and Salmonella isolated from poultry.
Materials and methods:
The essential oil was extracted using a Clevenger apparatus and subsequently its compounds were determined using GC-MS. Antibacterial properties of essential oil were determined by disc diffusion method, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). To evaluate the anti-biofilm properties the Microtiter plate test was used. Herbal essential oil was extracted and its compounds were identified correctly.
Results:
The major components of Satureja hortensis essential oil were thymol (41.28%), γ-terpinene (37.63%), p-cymene (12.2%) and α-terpinene (3.52%). The inhibition zone diameter in the disc diffusion test for E. coli and Salmonella were 32 ± 3 and 38 ± 4 mm, respectively, which was confirmed by MIC and MBC values. Regarding anti-biofilm activity, the MIC/2 concentration of S. hortensis significantly inhibited biofilm formation of E. coli. However, inhibition of biofilm formation of Salmonella was shown at concentration of MIC/2 and MIC/4.
Conclusion:
Based on our results, S. hortensis essential oil showed the growth inhibition and bactericidal activity against E. coli and Salmonella. Moreover, this study demonstrated anti-biofilm activity of S. hortensis essential against both tested bacteria.
Staphylococcus aureus (S. aureus) is the most common pathogen causing infections from skin to systemic infections. The success of S. aureus infections can partially be attributed to its antibiotic resistance and to its ability to form biofilm. An increasing prevalence of methicillin-resistant S. aureus (MRSA) becomes a global public health problem in recent decades. Here, the effects of tea catechin extracts on the growth and biofilm formation of three MRSA strains were investigated. The results revealed that tea catechin extracts potently suppressed MRSA growth, and the minimal inhibitory concentration of tea catechin extracts against these MRSA strains was 0.1 g/L. Then, tea catechin extracts inhibited biofilm formation of these strains in a dose-dependent manner measured with a colorimetric method, and the inhibitory effect was also demonstrated by scanning electron microscopy assay. Moreover, adhesin genes biofilm-associated protein (bap), bone sialoprotein-binding protein (bbp), collagen-binding protein (cna), clumping factors A (clfA), fibronectin binding protein A and B (fnbA and fnbB), and intercellular adhesion gene BC (icaBC) were scanned, and the results shown that fnbA and icaBC were present in these three strains. Furthermore, tea catechin extracts depressed fnbA and icaBC expression in the strains. Therefore, inhibition of biofilm formation by tea catechin extracts probably was associated with downregulation of fnbA and icaBC expression in these strains.
The opportunistic pathogen Staphylococcus aureus is responsible for causing infections related to indwelling medical devices, where this pathogen is able to attach and form biofilms. The intrinsic properties given by the self-produced extracellular biofilm matrix confer high resistance to antibiotics, triggering infections difficult to treat. Therefore, novel antibiofilm strategies targeting matrix components are urgently needed. The Biofilm Associated Protein, Bap, expressed by staphylococcal species adopts functional amyloid-like structures as scaffolds of the biofilm matrix. In this work we have focused on identifying agents targeting Bap-related amyloid-like aggregates as a strategy to combat S. aureus biofilm-related infections. We identified that the flavonoids, quercetin, myricetin and scutellarein specifically inhibited Bap-mediated biofilm formation of S. aureus and other staphylococcal species. By using in vitro aggregation assays and the cell-based methodology for generation of amyloid aggregates based on the Curli-Dependent Amyloid Generator system (C-DAG), we demonstrated that these polyphenols prevented the assembly of Bap-related amyloid-like structures. Finally, using an in vivo catheter infection model, we showed that quercetin and myricetin significantly reduced catheter colonization by S. aureus. These results support the use of polyphenols as anti-amyloids molecules that can be used to treat biofilm-related infections.
Vibrio parahaemolyticus and Escherichia coli are two major foodborne pathogens. In this paper, the antibiofilm activities of the ethanol extract of cinnamon against these two bacteria were studied in detail. The antibacterial activity and the MIC of the extract were determined, and the inhibition and removing effects of the extract on the biofilms of V. parahaemolyticus and E. coli were investigated. The biofilms stained with fluorescein isothiocyanate (FITC) and concanavalin A (Con A) were also observed by confocal laser scanning microscope (CLSM). The results indicated that the extract exhibited high antibacterial activity, with the MIC against V. parahaemolyticus and E. coli was 6.25 mg/mL. The effects on V. parahaemolyticus biofilm were significant with the inhibition rate of 75.46% at MIC, and the eradication rate of 93.26% at 32MIC, respectively. As to E. coli, the inhibition rate was 48.18% at MIC, and the eradication rate was 46.16% at 8MIC. Meanwhile, the extract could notably reduce the metabolic activities and the secretion of EPS in biofilm, it inhibited 78.57% EPS formation in V. parahaemolyticus biofilm at MIC, and eliminated 61.28% EPS in mature biofilm at 4MIC. CLSM images showed that the EPS of the treated biofilm became thinner and biofilm structure was looser, when compared with the untreated control. This study elucidated that the cinnamon extract was effective to prevent biofilm formation and eradicate mature biofilms of V. parahaemolyticus and E. coli.
Microorganisms, such as bacteria, tend to aggregate and grow on surfaces, secreting extracellular polymeric substances (EPS), forming biofilms. Biofilm formation is a life strategy, because through it microorganisms can create their own microhabitats. Whether for remediation of pollutants or application in the biomedical field, several methodological approaches are necessary for a more accurate analysis of the role and potential use of bacterial biofilms. The use of computerized microtomography to monitor biofilm growth appears to be an advantageous tool due to its non-destructive character and its ability to render 2D and 3D visualization of the samples. In this study, we used several techniques such as analysis of microbiological parameters and biopolymer concentrations to corroborate porosity quantified by 2D and 3D imaging. Quantification of the porosity of samples by microtomography was verified by increased enzymatic activity and, consequently, higher EPS biopolymer synthesis to form biofilm, indicating growth of the biofilm over 96h. Our interdisciplinary approach provides a better understanding of biofilm growth, enabling integrated use of these techniques as an important tool in bioremediation studies of environments impacted by pollutants.
The inhibitory and bactericidal activities of thyme oil against the foodborne multiple antibiotics-resistant Enterococcus faecalis biofilm were evaluated in this study. Gas chromatography-mass spectrometry revealed that more than 70% of the composition of thyme oil is thymol. Crystal violet staining assay showed that 128 and 256 μg/mL thyme oil significantly inhibited the biofilm formation of E. faecalis. The cell adherence of E. faecalis, as shown by its swimming and swarming motilities, was reduced by thyme oil. The exopolysaccharide (EPS) quantification assay showed that thyme oil inhibited the EPS synthesis in E. faecalis biofilms. The 3D-view observations through confocal laser scanning and scanning electron microscopy suggested that cell adherence and biofilm thickness were decreased in thyme oil-treated biofilms. Quantitative real-time analyses showed that the transcription of ebp and epa gene clusters, which were related to cell mobility and EPS production, was inhibited by thyme oil. Thus, thyme oil effectively inhibited the biofilm formation of E. faecalis by affecting cell adherence and EPS synthesis. Furthermore, 2048 and 4096 μg/mL thyme oil can effectively inactivate E. faecalis population in the mature E. faecalis biofilms by 5.75 and 7.20 log CFU/mL, respectively, after 30 min treatment. Thus, thyme oil at different concentrations can be used as an effective antibiofilm or germicidal agent to control E. faecalis biofilms.
Shikonin, an active ingredient of Lithospermum erythrorhizon, exerts anti-inflammatory and antibacterial effects, and promotes wound healing. We investigated whether shikonin stimulated gingival tissue wound healing in human gingival fibroblasts (hGF). In addition, we evaluated the effects of shikonin on the mitogen-activated protein kinase (MAPK) signaling pathway, which has an important role in wound healing. hGF were subjected to primary culture using gingiva collected from patients. The cells were exposed to/treated with Shikonin at concentrations ranging from 0.01 to 100 μM. The optimal concentration was determined by cell proliferation and migration assays. Type I collagen and fibronectin synthesis, the gene expression of vascular endothelial growth factor (VEGF) and FN, and the phosphorylation of Extracellular signal-regulated kinase (ERK) 1/2 were investigated. Identical experiments were performed in the presence of PD98059 our data suggest, a specific ERK 1/2 inhibitor. Shikonin significantly promoted hGF proliferation and migration. Shikonin (1 µM) was chosen as the optimal concentration. Shikonin promoted type I collagen and FN synthesis, increased VEGF and FN expression, and induced ERK 1/2 phosphorylation. These changes were partially suppressed by PD98059. In conclusion, Shikonin promoted the proliferation, migration, type I collagen and FN synthesis, and expression of VEGF and FN via ERK 1/2 signaling pathway in hGFs. Therefore, shikonin may promote periodontal tissue wound healing.
Escherichia coli O157:H7 (E. coli O157:H7) easily forms biofilms on pores and veins of plant, which poses a serious hazard to safety of ready-to-eat vegetables. Therefore, this study was designed to evaluate inhibition mechanism of clove oil on E. coli O157:H7 biofilm and apply it to vegetable for reduce risk of contamination. The results of crystal violet staining showed that clove oil could effectively remove biofilm. After treatment of biofilm with clove oil (2 MBIC), the results of resazurin method showed that bacterial metabolic activity decreased by 61.48%, and results of phenol-sulfuric acid method and Coomassie brilliant blue method showed that extracellular protein and polysaccharide content decreased by 62.76% and 47.5%, respectively. This indicates that clove oil can affect biofilm through above aspects. Finally, solid liposomes (SLPs) were prepared and applied to vegetables, which was first time that SLPs were used to remove biofilms. The results showed that clove oil SLPs exhibited excellent anti-biofilm activity at 4 °C and 25 °C without affecting senses and quality of vegetables. Therefore, the clove oil SLPs can exert a long-lasting antibacterial effect, effectively removing E. coli O157:H7 biofilm on vegetable surface, and prolonging the shelf life.
Bacterial biofilms are multicellular aggregates enclosed in a self-created biopolymer matrix. Biofilm-producing bacteria have become a great public health problem worldwide because biofilms enable these microorganisms to evade several clearance mechanisms produced by host and synthetic sources. Over the past years, different flavonoids including quercetin have engrossed considerable interest among researchers owing to their potential anti-biofilm properties. To our knowledge, there is no review regarding effects of quercetin towards bacterial biofilms, prompting us to summarize experimental evidence on its anti-biofilm properties. Quercetin inhibits biofilm development by a diverse array of bacterial pathogens such as Enterococcus faecalis, Staphylococcus aureus, Streptococcus mutans, Escherichia coli, and Pseudomonas aeruginosa. Prevention of bacterial adhesion, suppression of quorum-sensing pathways, disruption or alteration of plasma membrane, inhibition of efflux pumps, and blocking nucleic acid synthesis have been documented as major anti-biofilm mechanisms of quercetin. Overall, anti-biofilm activity of quercetin can open up new horizons in a wide range of biomedical areas, from food industry to medicine.
We, the authors, Editors and Publisher of the journal Artificial Cells, Nanomedicine, and Biotechnology, have retracted the following article:
Liushun Jia, Zhen Zhu, Hongbo Li & Yaofeng Li (2019) Shikonin inhibits proliferation, migration, invasion and promotes apoptosis in NCI-N87 cells via inhibition of PI3K/AKT signal pathway. Artificial Cells, Nanomedicine, and Biotechnology, 47(1), 2662–2669, DOI: 10.1080/21691401.2019.1632870
Since publication, the authors noticed that when they repeated the reported experiments as part of their further research, some of the data could not be replicated. As this directly impacts the validity of the reported results and conclusions the authors alerted the issue to the Editor and Publisher and all have agreed to retract the article to ensure the integrity of the scholarly record.
We have been informed in our decision-making by our policy on publishing ethics and integrity and the COPE guidelines on retractions.
The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.
Candidiasis is often associated with the formation of biofilms. Candida albicans biofilms are inherently resistant to many clinical antifungal agents and have increasingly been found to be the sources of C. albicans infections. Novel antifungal agents against C. albicans biofilms are urgently needed. The aim of this study was to investigate the effect of shikonin (SK) against C. albicans biofilms and to clarify the underlying mechanisms. XTT reduction assay showed that SK could not only inhibit the formation of biofilms but also destroy the maintenance of mature biofilms. In a mouse vulvovaginal candidiasis (VVC) model, the fungal burden was remarkably reduced upon SK treatment. Further study showed that SK could inhibit hyphae formation and reduce cellular surface hydrophobicity (CSH). Real-time reverse transcription-PCR analysis revealed that several hypha- and adhesion-specific genes were differentially expressed in SK-treated biofilm, including the downregulation of ECE1, HWP1, EFG1, CPH1, RAS1, ALS1, ALS3, CSH1 and upregulation of TUP1, NRG1, BCR1. Moreover, SK induced the production of farnesol, a quorum sensing molecule, and exogenous addition of farnesol enhanced the antibiofilm activity of SK. Taken together, these results indicated that SK could be a favorable antifungal agent in the clinical management of C. albicans biofilms.
Avian pathogenic Escherichia coli (APEC), collectively known as causative agent of extraintestinal infections, is an important cause of morbidity and mortality in poultry. Currently, quorum sensing (QS), biofilm formation and virulence factors are considered as novel prospective targets for antimicrobial therapy to control APEC invasion. In addition, inflammatory responses are also served as the major pathological features of APEC invasion. This study was aimed to explore the effect of baicalin on APEC and APEC-induced inflammatory responses. After treatment with baicalin, we mainly examined the AI-2 secretion, biofilm formation, expression of virulence genes of APEC, and the levels of inflammatory cytokines, as well as the expression of NF-κB pathway. Our results showed that baicalin significantly inhibited the QS via decreasing the AI-2 secretion, biofilm formation, and the expression of virulence genes of APEC such as LsrB, LsrK, LuxS, pfs, H-NS, fimA, fimB, fyuA, csgA, csgB, and rpoS. Moreover, baicalin significantly attenuated the release of lactate dehydrogenase (LDH), and the adhesion of APEC to chicken type II pneumocytes to reduce cell damage. Furthermore, baicalin also inhibited the expression of pro-inflammatory cytokines and NF-κB activation. Thus, our data revealed that baicalin could interfere with the quorum sensing, biofilm formation and virulence genes expression to relieve the APEC pathogenicity. Additionally, baicalin decreased the inflammatory responses of chicken type II pneumocytes induced by APEC. Taken together, these data provide a novel potential pharmaco-therapeutic approach to chicken colibacillosis.
Acinetobacter baumannii is a leading cause of nosocomial infections due to its increased antibiotic resistance and virulence. The ability of A. baumannii to form biofilms contributes to its survival in adverse environmental conditions including hospital environments and medical devices. A. baumannii has undoubtedly propelled the interest of biomedical researchers due to its broad range of associated infections especially in hospital intensive care units. The interplay among microbial physicochemistry, alterations in the phenotype and genotypic determinants, and the impact of existing ecological niche and the chemistry of antimicrobial agents has led to enhanced biofilm formation resulting in limited access of drugs to their specific targets. Understanding the triggers to biofilm formation is a step towards limiting and containing biofilm-associated infections and development of biofilm-specific countermeasures. The present review therefore focused on explaining the impact of environmental factors, antimicrobial resistance, gene alteration and regulation, and the prevailing microbial ecology in A. baumannii biofilm formation and gives insights into prospective anti-infective treatments.
Introduction: Biofilm formation is a strategy for microorganisms to adapt and survive in hostile environments. Microorganisms that are able to produce biofilms are currently recognized as a threat to human health.
Areas covered: Many strategies have been employed to eradicate biofilms, but several drawbacks from these methods had subsequently raised concerns on the need for alternative approaches to effectively prevent biofilm formation. One of the main mechanisms that drives a microorganism to transit from a planktonic to a biofilm-sessile state, is oxidative stress. Chemical agents that could target oxidative stress regulators, for instance antioxidants, could therefore be used to treat biofilm-associated infections.
Expert commentary: The focus of this review is to summarize the function and limitation of the current anti-biofilm strategies and will propose the use of antioxidants as an alternative method to treat, prevent and eradicate biofilms. Studies have shown that water-soluble and lipid-soluble antioxidants can reduce and prevent biofilm formation, by influencing the expression of genes associated with oxidative stress. Further in-vivo work should be conducted to ensure the efficacy of these antioxidants in a biological environment. Nevertheless, antioxidants are promising anti-biofilm agents, and thus is a potential solution for biofilm associated infections in the future.
Microtiter plate-based bacterial biofilm assay is frequently used to study bacterial biofilm development and growth. While this assay is simple and relatively high-throughput, it frequently shows difficulty in establishing robust biofilm attachment in the wells. We report that the consistency of bacterial biofilm assays carried out in microtiter plates subjected to abrasive treatment, by sandblasting or drill press grinding, is significantly improved in a Pseudomonas fluorescens Pf0-1 model. Scanning electron microscopy imaging suggests that the treated surfaces could provide points of attachment to facilitate the recruitment of bacteria in the initial phase of biofilm colony establishment. The sandblast treated polypropylene, but not polystyrene, plates were found suitable in studying the impact of flavonoid quercetin on the biofilm formation in Bacillus subtilis FB17. Further investigation revealed that due to the hydrophobicity of the polystyrene surfaces, a greater amount of quercetin was adsorbed on the plate surface, effectively lowering the concentration of the flavonoid in solution.
The present study aimed to understand the biofilm formation mechanism of Propionibacterium acnes by analyzing the components and structure of the biofilms. P. acnes strains were isolated from the surface of explanted cardiac pacemaker devices that exhibited no clinical signs of infection. Culture tests using a simple stamp culture method (pressing pacemakers against the surface of agar plates) revealed frequent P. acnes colonization on the surface of cardiac pacemaker devices. P. acnes was isolated from 7/31 devices, and the isolates were categorized by multilocus sequence typing into five different sequence types (STs): ST4 (JK18.2), ST53 (JK17.1), ST69 (JK12.2 and JK13.1), ST124 (JK5.3), ST125 (JK6.2), and unknown ST (JK19.3). An in vitro biofilm formation assay using microtiter plates demonstrated that 5/7 isolates formed biofilms. Inhibitory effects of DNase I and proteinase K on biofilm formation varied among isolates. In contrast, dispersin B showed no inhibitory activity against all isolates. Three-dimensional live/dead imaging of P. acnes biofilms with different biochemical properties using confocal laser microscopy demonstrated different distributions and proportions of living and dead cells. Additionally, it was suggested that extracellular DNA (eDNA) plays a role in the formation of biofilms containing living cells. Ultrastructural analysis of P. acnes biofilms using a transmission electron microscope and atmospheric scanning electron microscope revealed leakage of cytoplasmic components along with cell lysis and fibrous structures of eDNA connecting cells. In conclusion, the biochemical properties and structures of the biofilms differed among P. acnes isolates. These findings may provide clues for establishing countermeasures against biofilm-associated infection by P. acnes.
The quorum sensing (QS) circuit plays a role in the precise regulation of genes controlling virulence factors and biofilm formation in Pseudomonas aeruginosa. QS-controlled biofilm formation by Pseudomonas aeruginosa in clinical settings has remained controversial due to emerging drug resistance; therefore, screening diverse compounds for anti-biofilm or anti-QS activities is important. This study demonstrates the ability of sub-minimum inhibitory concentrations (sub-MICs) of baicalin, an active natural compound extracted from the traditional Chinese medicinal Scutellaria baicalensis, to inhibit the formation of Pseudomonas aeruginosa biofilms and enhance the bactericidal effects of various conventional antibiotics in vitro. In addition, baicalin exerted dose-dependent inhibitory effects on virulence phenotypes (LasA protease, LasB elastase, pyocyanin, rhamnolipid, motilities and exotoxin A) regulated by QS in Pseudomonas aeruginosa. Moreover, the expression levels of QS-regulatory genes, including lasI, lasR, rhlI, rhlR, pqsR and pqsA, were repressed after sub-MIC baicalin treatment, resulting in significant decreases in the QS signaling molecules 3-oxo-C12-HSL and C4-HSL, confirming the ability of baicalin-mediated QS inhibition to alter gene and protein expression. In vivo experiments indicated that baicalin treatment reduces Pseudomonas aeruginosa pathogenicity in Caenorhabditis elegans. Greater worm survival in the baicalin-treated group manifested as an increase in the LT50 from 24 to 96 h. In a mouse peritoneal implant infection model, baicalin treatment enhanced the clearance of Pseudomonas aeruginosa from the implants of mice infected with Pseudomonas aeruginosa compared with the control group. Moreover, the combination of baicalin and antibiotics significantly reduced the numbers of colony-forming units in the implants to a significantly greater degree than antibiotic treatment alone. Pathological and histological analyses revealed mitigation of the inflammatory response and reduced cell infiltration in the peritoneal tissue surrounding the implants after baicalin treatment. Measurement of the cytokine levels in the peritoneal lavage fluid of mice in the baicalin treatment group revealed a decrease in IL-4, an increase in interferon γ (IFN-γ), and a reversed IFN-γ/IL-4 ratio compared with the control group, indicating that baicalin treatment activated the Th1-induced immune response to expedite bacterial load clearance. Based on these results, baicalin might be a potent QS inhibitor and anti-biofilm agent for combating Pseudomonas aeruginosa biofilm-related infections.
The pathogenic bacterium Listeria monocytogenes can persist in food processing plants for many years, even when appropriate hygienic measures are in place, with potential for contaminating ready-to-eat products and, its ability to form biofilms on abiotic surfaces certainly contributes for the environmental persistence. In this research, L. monocytogenes was grown in biofilms up 8 days attached to stainless steel and glass surfaces, contributing for advancing the knowledge on architecture of mature biofilms, since many literature studies carried out on this topic considered only early stages of cell adhesion. In this study, biofilm populations of two strains of L. monocytogenes (serotypes 1/2a and 4b) on stainless steel coupons and glass were examined using regular fluorescence microscopy, confocal laser scanning microscopy and classic culture method. The biofilms formed were not very dense and microscopic observations revealed uneven biofilm structures, with presence of exopolymeric matrix surrounding single cells, small aggregates and microcolonies, in a honeycomb-like arrangement. Moreover, planktonic population of L. monocytogenes (present in broth media covering the abiotic surface) remained stable throughout the incubation time, which indicates an efficient dispersal mechanism, since the culture medium was replaced daily. In conclusion, even if these strains of L. monocytogenes were not able to form thick multilayer biofilms, it was noticeable their high persistence on abiotic surfaces, reinforcing the need to focus on measures to avoid biofilm formation, instead of trying to eradicate mature biofilms.
Infectious diseases continue to threaten human and animal health and welfare globally, impacting millions of lives and causing substantial economic loss. The use of antibacterials has been only partially successful in reducing disease impact. Bacterial cells are inherently resilient, and the therapy challenge is increased by the development of antibacterial resistance, the formation of biofilms and the ability of certain clinically important pathogens to invade and localize within host cells. Invasion into host cells provides protection from both antibacterials and the host immune system. Poor delivery of antibacterials into host cells causes inadequate bacterial clearance, resulting in chronic and unresolved infections. In this review, we discuss the challenges associated with existing antibacterial therapies with a focus on intracellular pathogens. We consider the requirements for successful treatment of intracellular infections and novel platforms currently under development. Finally, we discuss novel strategies to improve drug penetration into host cells. As an example, we discuss our recent demonstration that the cell penetrating cationic polymer polyhexamethylene biguanide has antibacterial activity against intracellular Staphylococcus aureus.
Linked articles:
This article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro-organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc.
Epigallocatechin-3-gallate (EGCG) is the major polyphenol in green tea. It has anti-microbial properties and disrupts the ordered structure of amyloid fibrils involved in human disease. The anti-microbial effect of EGCG against the opportunistic pathogen Pseudomonas aeruginosa has been shown to involve disruption of quorum sensing (QS). Functional amyloid fibrils in P. aeruginosa (Fap) are able to bind and retain quorum sensing molecules, suggesting that EGCG interferes with QS through structural remodeling of amyloid fibrils. Here we show that EGCG inhibits the ability of Fap to form fibrils; instead, EGCG stabilizes protein oligomers. Existing fibrils are remodeled by EGCG into non-amyloid aggregates. This fibril remodeling increases the binding of pyocyanin, demonstrating a mechanism by which EGCG can affect the QS function of functional amyloid. EGCG reduced the amyloid specific fluorescent Thioflavin T signal in P. aeruginosa biofilms at concentrations known to have exert an antimicrobial effect. Nanoindentation studies showed that EGCG reduced the stiffness of biofilm containing Fap fibrils but not in biofilm with little Fap. In a combination treatment with EGCG and tobramycin, EGCG had a moderate effect on the minimum bactericidal eradication concentration against wildtype P. aeruginosa biofilms while EGCG had a more pronounced effect when Fap were overexpressed. Our results provide a direct molecular explanation for EGCG's ability to disrupt P. aeruginosa's QS and modify its biofilm, and strengthens the case for EGCG as a candidate in multidrug treatment of persistent biofilm infections.
Bacterial biofilms are formed by communities that are embedded in a self-produced matrix of extracellular polymeric substances (EPS). Importantly, bacteria in biofilms exhibit a set of 'emergent properties' that differ substantially from free-living bacterial cells. In this Review, we consider the fundamental role of the biofilm matrix in establishing the emergent properties of biofilms, describing how the characteristic features of biofilms-such as social cooperation, resource capture and enhanced survival of exposure to antimicrobials-all rely on the structural and functional properties of the matrix. Finally, we highlight the value of an ecological perspective in the study of the emergent properties of biofilms, which enables an appreciation of the ecological success of biofilms as habitat formers and, more generally, as a bacterial lifestyle.
Introduction:
The role of natural bioactive substances in treating infections has been rediscovered as bacterial resistance become common to most of the antibiotics. Curcumin is a bioactive substance from turmeric. Owing to antimicrobial properties, its prospect as an antibacterial agent is currently under focus.
Materials and methods:
We have evaluated the in vitro synergy of curcumin with antibiotics against sixty biofilm producing bacterial isolates. Congo red agar method was used to identify the biofilm producing isolates. Curcumin minimum inhibitory concentration (MIC) was determined by agar dilution method. Its antibiotic synergy was identified by the increase in disc diffusion zone size on Mueller-Hinton agar with 32 mg/L curcumin.
Results:
The mean MICs of curcumin against Gram-positive and Gram-negative isolates were 126.9 mg/L and 117.4 mg/L, respectively. Maximum synergy was observed with ciprofloxacin among Gram-positive and amikacin, gentamicin, and cefepime among Gram-negative isolates.
Conclusions:
Curcumin per se as well as in combination with other antibiotics has a demonstrable antibacterial action against biofilm producing bacterial isolates. It may have a beneficial role in supplementing antibiotic therapy.
Pseudomonas aeruginosa infections result in high morbidity and mortality rates for individuals with cystic fibrosis (CF), with premature death often occurring. These infections are complicated by the formation of biofilms in the sputum. Antibiotic therapy is stymied by antibiotic resistance of the biofilm matrix, making novel antibiofilm strategies highly desirable. Within P. aeruginosa biofilms, the redox factor pyocyanin enhances biofilm integrity by intercalating with extracellular DNA. The antioxidant glutathione (GSH) reacts with pyocyanin, disrupting intercalation. This study investigated GSH disruption by assaying the physiological effects of GSH and DNase I on biofilms of clinical CF isolates grown in CF artificial sputum medium (ASMDM+). Confocal scanning laser microscopy showed that 2 mM GSH, alone or combined with DNase I, significantly disrupted immature (24-h) biofilms of Australian epidemic strain (AES) isogens AES-1R and AES-1M. GSH alone greatly disrupted mature (72-h) AES-1R biofilms, resulting in significant differential expression of 587 genes, as indicated by RNA-sequencing (RNA-seq) analysis. Upregulated systems included cyclic diguanylate and pyoverdine biosynthesis, the type VI secretion system, nitrate metabolism, and translational machinery. Biofilm disruption with GSH revealed a cellular physiology distinct from those of mature and dispersed biofilms. RNA-seq results were validated by biochemical and quantitative PCR assays. Biofilms of a range of CF isolates disrupted with GSH and DNase I were significantly more susceptible to ciprofloxacin, and increased antibiotic effectiveness was achieved by increasing the GSH concentration. This study demonstrated that GSH, alone or with DNase I, represents an effective antibiofilm treatment when combined with appropriate antibiotics, pending in vivo studies.
Terpenes, a large family of natural products with important applications, are commonly associated with plants and fungi. The diterpenoids dehydroabietic and abietic acids are defense metabolites abundant in resin, and are used as biomarkers for conifer plants. We report here for the first time that the two diterpenoid acids are produced by members of several genera of cyanobacteria. Dehydroabietic acid was isolated from two cyanobacterial strains and its identity was confirmed spectroscopically. One or both of the diterpenoids were detected in the cells of phylogenetically diverse cyanobacteria belonging to four cyanobacterial 'botanical orders', from marine, estuarine and inland environments. Dehydroabietic acid was additionally found in culture supernatants. We investigated the natural role of the two resin acids in cyanobacteria using ecologically-relevant bioassays and found that the compounds inhibited the growth of a small coccoid cyanobacterium. The unexpected discovery of dehydroabietic and abietic acids in a wide range of cyanobacteria has implications for their use as plant biomarkers.
The development of drug-resistant bacteria as a result of the inappropriate use of antibiotics, has led to a study on other sources of drugs for the treatment of bacterial infections. Natural compounds derived from medicinal plants have long been considered a rich source of therapeutic agents. Scutellaria baicalensis has attracted attention as its root extracts are used for a variety of therapeutic purposes. Published papers involving Scutellaria baicalensis in Scopus, PubMed, and Google Scholar databases were collected and annotated for their antimicrobial and antibiofilm application. Baicalin is the most important compound derived from the roots of Scutellaria baicalensis, and it has antimicrobial, antioxidant, anticancer, and anti-inflammatory properties that can be used for the treatment of several diseases. The main active compounds were identified as natural antimicrobial and anti-biofilm agents with clear-cut mechanisms or identified molecular reports. The present review summarizes the mechanisms of biofilm formation and quorum sensing, as well as recent advances in the discovery and identification of plant-derived natural products which are antimicrobial and anti-biofilm agents. In addition, clinical trials involving plant-derived anti-biofilm therapeutics were also discussed. These newly discovered naturally occurring anti-biofilm agents are promising candidates that could provide novel strategies for combating pathogenic bacteria and treating biofilm-associated infections.
Shikonin is a bioactive compound found in the roots of Lithospermum erythrorhizon and has demonstrated antibacterial activity. In the current study, we aimed to examine the antibiofilm activity of shikonin against Listeria monocytogenes, and to investigate its effects on key L. monocytogenes virulence factors associated with human infection. Present results showed that the minimum inhibitory concentration of shikonin against L. monocytogenes ranged from 25 to 100 μg/mL. Crystal violet staining confirmed that sub-inhibitory concentrations (SICs) of shikonin significantly reduced biofilm biomass in as little as 24 h. Decreased biofilm adhesion, altered biofilm morphology, and disruption of biofilm architecture following treatment with SICs of shikonin were also confirmed by light microscopy and field-emission scanning electron microscopy. Further, at SICs, shikonin effectively reduced the ability of L. monocytogenes to adhere to and invade HT-29 cells and prevented the secretion of listeriolysin O. Moreover, the transcription of biofilm-associated genes and key virulence genes that contribute to infection was also suppressed by shikonin at SICs. Therefore, present results suggest that shikonin could be used as an alternative antibacterial agent to combat L. monocytogenes biofilm formation and infection.
Porphyromonas gingivalis is a keystone pathogen and major colonizer in host tissue which plays a pivotal role in periodontitis among the other polymicrobial infections. Increasing facts demonstrate that curcumin has antibacterial activity and anti-biofilm effect against the periodontopathogens through diverse mechanisms that have a positive impact on periodontal health. The present study was aimed to elucidate the effect of curcumin on biofilm formation and virulence factor gene expression of P. gingivalis. By using gene expression studies, we exploited the mechanism of anti-biofilm effects of curcumin on P. gingivalis. The minimum inhibitory concentration and minimum bactericidal concentration of curcumin for both ATCC and clinical strains of P. gingivalis were found to be 62.5 and 125 µg ml−1 respectively. Curcumin prevented bacterial adhesion and biofilm formation in a dose-dependent manner. Further, curcumin attenuated the virulence of P. gingivalis by reducing the expression of genes coding for major virulence factors, including adhesions (fimA, hagA, and hagB) and proteinases (rgpA, rgpB, and kgp). The results indicated that curcumin has shown anti-biofilm as well as antibacterial activity against P. gingivalis. Further, curcumin because of its pleiotropic actions could be a simple and inexpensive therapeutic strategy in the treatment of periodontal disease.
Vibrio mimicus is a relatively rare food-borne pathogen in seafood and water. Rare reports have been published to investigate the biofilm of Vibrio mimicus. (−)-epigallocatechin-3-gallate (EGCG), the major polyphenolic component of tea, can interfere with bacterial biofilm formation. This study showed that Vibrio mimicus was capable of forming high amounts of biofilms in various culture media. Sub-MICs of EGCG significantly reduced the biofilm production of Vibrio mimicus at 15 °C, 28 °C and 37 °C. Confocal laser-scanning microscope (CLSM) observation proved that the architecture of Vibrio mimicus biofilm was affected by EGCG at the concentration of 64 μg/mL (1/4 MIC). EGCG reduced Vibrio mimicus autoaggregation and swimming motility. EGCG also increased membrane permeability and ROS production, caused cell membrane damage and led to potassium leakage. These may contribute to the antibiofilm efficacy of EGCG against Vibrio mimicus. Our work showed the inhibitory effects of sub-MICs of EGCG on Vibrio mimicus biofilm for the first time, supporting the potential application of EGCG as a natural antibiofilm agent in the food industry.
Essential oil extracted from eucalyptus leaves has a long folkloric history of being used for the treatment of respiratory conditions. The primary terpenoid constituent in the essential oil of eucalyptus (EOE) is eucalyptol (1,8-cineole or cineole), which has been studied in both preclinical and clinical settings. Eucalyptol displays several pharmacologic activities that may provide therapeutic effects in respiratory conditions such as anti-inflammatory and bronchodilatory effects. Several clinical trials have been performed in persons affected with respiratory illness such as rhinosinusitis, bronchitis, asthma and chronic obstructive pulmonary disorder (COPD) with positive results. The objectives of this review are to provide a mechanistic overview of EOE with a focus on the primary constituent eucalyptol, summarize the pharmacology of eucalyptol in respiratory conditions, describe clinical trial data that has been generated by studying eucalyptol in humans for respiratory conditions, and discuss clinical and safety considerations for the use of EOE or eucalyptol.
In recent years, studies have found that in addition to nosocomial infections, methicillin-resistant Staphylococcus aureus (MRSA) can even cause community-associated infections, and MRSA spreads through food and animal that produce products in the community. In addition, MRSA can colonize the surface of food and food container and form biofilms by secreting extracellular polymers, leading to cross-infection. Cardamom essential oil has been found to be highly active against MRSA. Therefore, this study aimed to investigate the inhibitory effect of cardamom essential oil on MRSA biofilm and its mechanism. The semi-quantitative test results of crystal violet and the removal of MRSA biofilm on different food container materials show that cardamom essential oil can effectively remove MRSA biofilm and can be applied to the surface of food container materials. Meanwhile, the inhibition mechanism of cardamom essential oil on MRSA biofilm was studied by measuring the metabolic activity, extracellular polymer content and relative expression of MRSA biofilm formation related genes. Based on results, it is presumed that cardamom essential oil can function to scavenge MRSA biofilm by inhibiting the metabolic activity of bacteria, inhibiting the formation of extracellular polymers, and regulating the expression of genes involved in biofilm formation.
Dental caries is a type of oral microbiome dysbiosis and biofilm infection that affects oral and systemic conditions. For healthy life expectancy, natural bacteriostatic products are ideal for daily and lifetime use as anti-oral infection agents. This study aimed to evaluate the inhibitory effects of abietic acid, a diterpene derived from pine rosin, on the in vitro growth of cariogenic bacterial species, Streptococcus mutans. The effective minimum inhibitory concentration of abietic acid was determined through observation of S. mutans growth, acidification, and biofilm formation. The inhibitory effects of abietic acid on the bacterial membrane were investigated through the use of in situ viability analysis and scanning electron microscopic analysis. Cytotoxicity of abietic acid was also examined in the context of several human cell lines using tetrazolium reduction assay. Abietic acid was found to inhibit key bacterial growth hallmarks such as colony forming ability, adenosine triphosphate activity (both planktonic and biofilm), acid production, and biofilm formation. Abietic acid was identified as bacteriostatic, and this compound caused minimal damage to the bacterial membrane. This action was different from that of povidone-iodine or cetylpyridinium chloride. Additionally, abietic acid was significantly less cytotoxic compared to povidone-iodine, and it exerted lower toxicity towards epithelial cells and fibroblasts compared to that against monocytic cells. These data suggest that abietic acid may prove useful as an antibacterial and antibiofilm agent for controlling S. mutans infection.
In recent years there has been a dramatic increase in human exposure to various genotoxic agents, such as ultraviolet light, ionizing radiation, or air, water and food pollutants. Continuous impaired genomic stability is known to cause the mutations that are actively involved in many pathological processes including cancer. A wide array of phytochemicals have been reported to exert genoprotective effects that are mainly related to their antioxidant potential. This chapter discusses the antigenotoxic properties of some nonphenolic phytochemicals, such as organosulfur compounds, terpenoids, and polysaccharides. It does so in terms of study type, mechanisms of activity, structure–activity relationships, and their clinical significance.
Enterococcus faecalis is a gram positive enteric commensal bacteria or opportunistic pathogen and its infection involves biofilm formation. Quercetin, a plant origin polyphenol was found to inhibit E. faecalis biofilm. Crystal violet assay, SEM and CLSM microscopy confirmed biofilm inhibition by quercetin. Proteomics was used to elucidate the changes occurred in bacterial cell by quercetin treatment. 2D-Electrophorosis and MALDI-TOF analysis revealed that nineteen proteins were differentially expressed in quercetin treated sample. Glycolytic pathways, protein translation-elongation pathways and protein folding pathways were under differential expression after treatment. Real Time-PCR (RT-PCR) validated the proteomic data at genomic level except for the translation elongation factor G which showed opposite data to proteomics. Protein-protein interaction networks constructed using STRING 10.0 demonstrated strong connection of translation-elongation proteins with many important proteins. The results of the comparative analysis indicate that quercetin exerts its inhibitory effect by disturbing glycolytic, protein translation-elongation and protein folding pathways. This disturbs bacterial physiology and stops transition of planktonic cells to biofilm state.
Upon single treatment against Staphylococus aureus, quercetin–pivaloxymethyl conjugate (Q-POM) had antibacterial activities with minimum inhibitory concentrations (MICs) of 16–32 mg/L. Q-POM showed MIC of 32 mg/L against vancomycin-resistant Enterococcus faceium (VRE), which is remarkably lower than other antibiotics investigated (≥256 mg/L). Under sub-MIC concentrations, Q-POM potentiated the activity of ampicillin, cefepime, and vancomycin against S. aureus and Enterococcus (including highly resistant strains such as hetero-resistant vancomycin-intermediate S. aureus (hVISA), vancomycin-intermediate S. aureus (VISA), and VRE), by decreasing the MICs of these antibiotics by 4–128 folds. Q-POM was found to be partially synergistic with ampicillin and cefepime against S. aureus and Enterococcus, while it was strongly synergistic with vancomycin. Q-POM at 5 mg/L inhibited the formation of biofilms of S. aureus by 24–83% and VRE by 70%. Additionally, Q-POM inhibited the hemolytic activity of S. aureus in a dose-dependent manner. Cytotoxic activity was evaluated in human liver epithelial cells (HepG2), and the 50% cytotoxicity concentration (CC50) value of Q-POM was higher than 50 mg/L. These results indicate the potential use of Q-POM in treatment of methicillin-resistant Staphylococcus aureus (MRSA) and VRE infections.
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The aim of the present study was to investigate the inhibitory mechanisms of quercetin against L. monocytogenes biofilm formation, by affecting secretion of polymeric substances (EPS) and spatio-temporal organization. The minimum concentration of quercetin inhibiting the establishment of bacterial biofilm was determined to be 0.8 mM; therefore, a lower concentration of the flavonoid (0.2 mM) was used in order to investigate its effect on cell attachment and EPS production on stainless steel and the spatio-temporal organization on glass, while avoiding bias of loss of cellular viability. In applying this concentration, reductions of 1.32 and 1.94 Log10 CFU/cm2 of initial adhesion and maximum cell density on stainless steel were observed on the test surfaces at 2 h (initial adhesion) and 24 h (maximum cell density), respectively. EPS on mature biofilms (24 h) was mainly composed by proteins (97.83 %), followed by polysaccharides (2.03 %) and DNA (0.14 %). In addition, quercetin reduced total extracellular protein content by 41% compared with control; while, DNA and polysaccharide contents in biofilms were not affected. Also, relative to control, quercetin impaired surface colonization, early stages of biofilm formation and the development of a more complex architecture during its formation. This proved that quercetin inhibited the formation of L. monocytogenes biofilms by reducing initial attachment, the amount of extracellular proteins found in the EPS matrix and the overall spatio-temporal organization of L. monocytogenes.
Listeria monocytogenes (L. monocytogenes) is a well-known food-borne pathogen that causes systemic listeriosis. Its biofilm-forming ability is known to be important for its antimicrobial resistance and persistence. Epigallocatechin-gallate (EGCG) is the highest component of tea polyphenols in tea extracts and has broad-spectrum antimicrobial activities. In this study, the efficacys of EGCG to inhibit biofilm formation and hemolytic activity of L. monocytogenes were determined. EGCG at 20μg/mL, 40μg/mL(less than compound’s minimum inhibitory concentration, MIC) and 200 μg/mL (1 MIC) were tested. Crystal violet staining showed that sub-MIC and MIC of EGCG could significantly reduce the biofilm formation by L. monocytogenes on polystyrene microtiter plates at three temperatures (15°C, 30°C and 37°C). EGCG reduced the sessile cell numbers present in biofilm at those temperatures. EGCG also significantly inhibited hemolytic activity of L. monocytogenes as measured by sheep red blood cells. Real time PCR assay was used to investigate the relative gene expression of L. monocytogenes grown at 37°C and revealed that EGCG down-regulated virulence genes (inlA and hly), SOS response genes (recA and yneA) and quorum sensing gene (agrA). These results suggested the feasibility of using EGCG in food industry to control L. monocytogenes biofilm formation.
Several pathogens associated with chronic infections, including Pseudomonas aeruginosa in cystic fibrosis pneumonia, Haemophilus influenzae and Streptococcus pneumoniae in chronic otitis media, Staphylococcus aureus in chronic rhinosinusitis and enteropathogenic Escherichia coli in recurrent urinary tract infections, are linked to biofilm formation. Biofilms are usually defined as surface-associated microbial communities, surrounded by an extracellular polymeric substance (EPS) matrix. Biofilm formation has been demonstrated for numerous pathogens and is clearly an important microbial survival strategy. However, outside of dental plaques, fewer reports have investigated biofilm development in clinical samples. Typically biofilms are found in chronic diseases that resist host immune responses and antibiotic treatment and these characteristics are often cited for the ability of bacteria to persist in vivo. This review examines some recent attempts to examine the biofilm phenotype in vivo and discusses the challenges and implications for defining a biofilm phenotype
Two new iridoid type monoterpenoids, named syringopicrogenin D (1) and syringopicrogenin E (2), were isolated from the EtOH extract of the fresh leaves of Syringa oblate Lindl. The structures of 1 and 2 were elucidated by analyzing spectroscopic data and comparing their NMR data with those of the previously reported compound 1a.
Propionibacterium acnes is an opportunistic pathogen which has become notorious owing to its ability to form a recalcitrant biofilm and to develop drug resistance. The current study aimed to develop anti-biofilm treatments against clinical isolates of P. acnes under in vitro and in vivo conditions. A combination of ellagic acid and tetracycline (ETC; 250 μg ml(-1) + 0.312 μg ml(-1)) was determined to effectively inhibit biofilm formation by P. acnes (80-91%) without affecting its growth, therefore potentially limiting the possibility of the bacterium attaining resistance. In addition, ETC reduced the production of extracellular polymeric substances (EPS) (20-26%), thereby making P. acnes more susceptible to the human immune system and antibiotics. The anti-biofilm potential of ETC was further substantiated under in vivo conditions using Caenorhabditis elegans. This study reports a novel anti-biofilm combination that could be developed as an ideal therapeutic agent with broad cosmeceutical and pharmaceutical applicability in the era of antibiotic resistance.
Biofilms constitute the predominant microbial style of life in natural and engineered ecosystems. Facing harsh environmental conditions, microorganisms accumulate reactive oxygen species (ROS), potentially encountering a dangerous condition called oxidative stress. While high levels of oxidative stress are toxic, low levels act as a cue, triggering bacteria to activate effective scavenging mechanisms or to shift metabolic pathways. Although a complex and fragmentary picture results from current knowledge of the pathways activated in response to oxidative stress, three main responses are shown to be central: the existence of common regulators, the production of extracellular polymeric substances, and biofilm heterogeneity. An investigation into the mechanisms activated by biofilms in response to different oxidative stress levels could have important consequences from ecological and economic points of view, and could be exploited to propose alternative strategies to control microbial virulence and deterioration.